This invention relates to a method of identifying genetically modified cells using a mutated protein-tyrosine kinase receptor (PTKR), particularly a mutated epidermal growth factor receptor (EGFR) family member or a mutated muscle specific kinase (MuSK) family member as a selectable cell marker.
The use of selectable markers is well known for the identification of prokaryotic and eukaryotic cells, and the use is essential because frequently when a DNA sequence of interest is introduced into a cell it will not necessarily lead to a phenotype that is readily determined. The number of selectable markers used in identifying eukaryotic cells and especially mammalian cells has been limited. In the past, selectable markers that conferred drug resistance were employed (i.e. G-418 and hygromycin). More recently, selectable markers that are combined with fluorescence activated cell sorting (FACS) have been used, for example, green fluorescent protein (GFP). Alternatively, antibodies that recognize a cell surface molecule may be coupled to a fluorophore to help identify the cells of interest.
Several cell surface molecules have been used as a selectable cell marker including murine CD8, CD24, and human Low-Affinity Nerve Growth Factor Receptor (NGFR). Reference is made to the following publications; WO95/06723; WO98/19540; Jolly, et al., Proc. Natl. Acad. Sci. 80: 477 (1983); and Reddy, et al., Mol. Brain Res. 8:137 (1990). Some of these cell surface molecules have been mutated in their intracellular domain to avoid signaling of the molecule when binding to their ligand. However upon ligand binding some of the intracellularly mutated molecules may homo-, heterodimerize or trimerize. If a newly introduced molecule in the cell should heterodimerize with endogenous receptors a dominant negative effect may result. The present invention provides cell surface markers that do not heterodimerize with endogenous receptors.
The present invention provides a method of identifying genetically modified mammalian cells comprising introducing a nucleic acid sequence encoding a mutated protein-tyrosine kinase receptor (PTKR) operatively linked to an expression control sequence into a cell to form a genetically modified cell; allowing expression of the mutated PTKR in the genetically modified cell; and identifying said genetically modified cell expressing the mutated PTKR. In a preferred embodiment the mutated PTKR is a mutated epidermal growth factor receptor (EGFR) family member (or MuSK family member), particularly preferred is a mutated EGFR1 and more specifically the sequences given the designation EGFR1-I and EGFR1-II. In a second embodiment the introducing step is accomplished by incorporating the nucleic acid sequence encoding the mutated PTKR, and particularly the mutated EGFR into a vector and introducing the vector into a cell. The preferred vectors are retroviral vectors. In a further embodiment the mammalian cells are human cells, particularly hematopoietic cells, liver cells, endothelial vascular cells, and smooth muscle cells, more particularly hematopoietic cells. In yet another embodiment a beterologous gene is incorporated into the construct or vector comprising the mutated PTKR marker sequence. In yet a further embodiment the identifying step is accomplished by contacting the genetically modified cells with an antibody that recognizes and binds to the mutated PTKR. In yet another embodiment the identifying step separates the genetically modified cells from the non-genetically modified cells.
In a second aspect, the invention provides a method of identifying genetically modified mammalian cells comprising the steps of incorporating into a vector a nucleic acid sequence encoding a mutated epidermal growth factor receptor (EGFR) family member operatively linked to an expression control sequence; introducing the vector into a mammalian cell to form a genetically modified cell; allowing expression of the mutated EGFR in the genetically modified cell; and identifying said genetically modified cell expressing the mutated EGFR. In a preferred embodiment the mutated EGFR is EGFR1-I or EGFR1-II and the vector is a retroviral vector.
In a third aspect, the invention is directed to a method for the immunoselection of transduced mammalian cells comprising, retrovirally transducing mammalian cells with a nucleic acid sequence encoding a mutated epidermal growth factor receptor (EGFR) family member operatively linked to an expression control sequence; incubating the transduced cells with a marked antibody that recognizes and binds specifically to the mutated EGFR; and identifying the marked transduced cells. In a preferred embodiment the cells are human cells, particularly hematopoietic cells. In another embodiment the cells are transduced by a retroviral vector derived from the group consisting of moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV) and spleen focus forming virus (SFFV). In a further embodiment the cells are transduced with a vector derived from a lentivirus. In yet a further embodiment the method includes the step of separating the identified marked transduced cells from non-marked cells. In yet another embodiment the method includes the step of expanding the marked transduced cells.
In a fourth aspect, the invention is directed to a method of identifying mammalian cells expressing a protein of interest, comprising the steps of, introducing into a mammalian cell a nucleic acid sequence encoding a mutated PTKR operatively linked to an expression control sequence and a nucleic acid sequence encoding a protein of interest; culturing the resulting mammalian cells; and identifying cells which express the mutated PTKR thereby obtaining cells which express the protein of interest.